Nicotinamide nucleotides play a major role in biological oxidations; reductive synthesis of carbohydrates, lipids and amino acids; the various monooxygenase reactions involved in detoxification and steroid hydroxylation reactions; energy production; and metabolic regulation. Nicotinamide nucleotide transhydrogenases are potential control devices for maintaining the cellular balance of NAD(H) and NADP(H), and thereby contributing to the regulation of various metabolic reactions linked to these nucleotides. The mitochondrial transhydrogenase system is of particular interest, because the equilibrium of the mitochondrial nicotinamide nucleotide pool can be shifted in favor of NADPH production in an endergonic reaction, which utilizes stoichiometric amounts of ATP. The objectives of the program are (1) to study the molecular mechanisms of nonenergy-linked (NAD(P)H yields NAD) and energy-linked (NAD(P)H yields NADP) transhydrogenation; (2) to isolate and characterize the enzymes involved; (3) to investigate the mechanisms of energy transfer to, and utilization by, the transhydrogenase enzyme; and (4) as a long-term goal to relate these findings to studies on (a) the general mechanism of energy conservation, transduction and transfer by mitochondria, and (b) the physiological role of the mitochondrial transhydrogenase reaction. NADH dehydrogenase, which is capable of NAD(P)H yields NAD transhydrogenation has been purified, and its molecular and enzymatic properties studied (Y.M. Galante and Y. Hatefi (1979) Arch. Biochem. Biophys. 192, 559-568). Mechanism of energy-linked transhydrogenation has been studied, using NAD(P)H yields Acetylpyridine- or thionicotinamide-NADP transhydrogenations under energy-linked and nonenergy-linked conditions. The Vmax and Km values as a function of pH (5.5 to 8.5) have indicated that acidification of assay medium mimics the kinetics of energy-linked reaction by increasing enzyme-substrate affinity.